DESCRIPTION (from applicant's abstract): Enkephalin and beta-endorphin opioid peptide neurotransmitters are synthesized as proenkephalin (PE) and proopiomelanocortin (POMC) precursors, respectively, which require proteolytic processing in secretory vesicles to form active neuropeptides. The extent of PE and POMC processing in chromaffin cells and pituitary, respectively, is limited and varied. These observations suggest that endogenous protease inhibitors may be involved in proneuropeptide processing. Therefore, the investigator plans to investigate the possibility that endogenous protease inhibitors may be colocalized with, and inhibit, pro-neuropeptide processing enzymes. The investigator's molecular studies have identified two novel, secretory vesicle protease inhibitors, endopin 1 and 2 that possess distinct target protease specificities. These two endopins share homology with the serpin family of protease inhibitors, and possess active sites consistent with pro-neuropeptide processing. Endopins are colocalized with, and inhibit the PE processing enzyme PTP ('prohormone thiol protease'). To expand these studies the goal of this proposal is to compare the roles of endopins 1 and 2 in the proteolytic processing of the pro-neuropeptides PE and POMC. In specific aim 1, the target protease specificities of endopin 1and 2 are to be evaluated with classical proteases (trypsin and others), and pro-neuropeptide processing enzymes involved in processing PE and POMC (PTP and PC enzymes) will be studied. Active-site mutants and chimeras of endopins will define structural features responsible for inhibition. In specific aims 2 and 3, regulation of PE and POMC processing of endopins are to be examined in primary cultures of chromaffin and pituitary cells, respectively. The roles of endopins in PE and POMC processing are to be compared utilizing endopin sense and antisense expression. Also, the tissue distribution of the endopins are to be studied in specific aim 4. Recent genetic studies indicate that new proteases critical for POMC processing in the anterior pituitary have yet to be identified. Because secretory vesicle endopins (present in the anterior pituitary) form complexes with proteases, endopins can be used as a tool for expression cloning of endopin-interacting clones from the anterior pituitary (aim 5). Evaluation of an endopin-interacting clone for enhanced POMC processing, regulated secretion, and inhibition by endopin(s), will lead to consideration of the clone as an endopin target protease for POMC processing.